Restrictocin-like ribotoxin analogues comprising only one cysteine available for covalent linkage to a partner

ABSTRACT

Ribotoxins, such as restrictocin, mitogillin and alpha-sarcin which do not contain a cysteine for linkage to antibodies for immunotoxin production need to be modified for such linkage without compromising biological activity. Protein analogues of such native ribotoxins are provided in which both the analogue and the native ribotoxin can cleave only a single phosphodiester bond of 28S rRNA in a 60S ribosomal subunit and in which the analogue comprises only one cysteine available from covalent linkage to a partner, such as an antibody, which cysteine is not present in the native ribotoxin. Especially preferred analogues are restrictocin Cys 13, Cys 82, Cys 106 and Cys 110 and restrictocin Cys 150-Gly151. Corresponding DNA sequences, vectors, host cells, immunotoxins and pharmaceutical compositions are also claimed.

BACKGROUND OF THE INVENTION

Ribotoxins are potent inhibitors of protein synthesis. They are believedto act as enzymes on the 28S rRNA of the eucaryotic ribosome and can bedivided into two classes (Jiminez, A et al., 1985, Ann. Rev Microbiol.,39, 649-672, and Wool, I. G. et al., 1990, in Hill, W. E. et al. (ed.)The Ribosome Structure, Function & Evolution, American Society ofMicrobiology, Washington D.C., 203-214). Restrictocin, mitogillin andα-sarcin belong to the class which cleaves a single phosphodiester bond.Ricin belongs to the other class which cleaves an N-glycosidic linkagebetween base and ribose. The nucleotide sequence of the restrictocingene has been described by Lamy, B. & Davies, J. in Nucleic AcidsResearch, 1991, 19, 1001-1006.

Immunotoxins are an example of a conjugate formed between two partnersnamely a toxin and an immunoglobulin or antibody. The partners areusually joined via a chemical linker but may be joined directly.Ribotoxins such as for example restrictocin may be used as the toxincomponent of immunotoxins, linked to the antibody via a variety ofchemical linkers. Known methods involve derivatisation of therestrictocin with chemicals such as iminothiolane (although otherderivatising agents, eg. N-succinimidyl-3-(2-pyridyldithio)propionate(hereinafter abbreviated SPDP) may be used) to introduce a sulphydrylfunctionality and to form a disulphide cross-linked structure with anappropriately derivatised antibody.

Due to the inherent random nature of the chemical derivatisation at,primarily (although not necessarily solely), amino groups such as forexample on the restrictocin molecule of which there are 17 including thealpha amino group, a heterogeneous product results comprisingunderivatised restrictocin, mono, di, tri etc derivatised moleculeswhich may result in a complex product profile following conjugation toantibody (1,2,3 etc moles of restrictocin/mole antibody or, potentially,1,2,3 etc moles of antibody/mole Restrictocin). Such a heterogeneousproduct undesirably produces batch to batch variability and reducedoverall yield. Batch to batch variability is particularly undesirable inthe pharmaceutical field where the product may be for example animmunotoxin wherein the antibody is selective for tumours and is used incancer therapy.

In addition chemical derivatisation may derivatise residues close to theactive site of the molecule so compromising biological activity of thederivatised toxin.

SUMMARY OF THE INVENTION

The present invention seeks to overcome these disadvantages by providinga protein analogue having a suitable sulphydryl functionality such thata specific amino acid residue in the protein sequence provides a singleconjugation site at a defined position.

According to one aspect of the present invention there is provided aprotein analogue of a native ribotoxin in which both the proteinanalogue and the native ribotoxin can cleave only a singlephosphodiester bond of 28S rRNA in a 60S ribosomal subunit and in whichthe protein analogue comprises only one cysteine available for covalentlinkage to a partner said cysteine not being present in the nativeribotoxin.

The term "can cleave a single phosphodiester bond of 28S rRNA in a 60Sribosomal subunit" as used herein relates to the extremely selectiveaction of the class of ribotoxins typified by restrictocin, mitogillinand alpha-sarcin (Wool, I. G., Jan 1984, Trends Biochem. Sci. 14-17).Thus protein analogues of the present invention substantially retain thebiological activity of ribotoxins such as restrictocin, mitogillin andalpha-sarcin. The present invention thus further discloses sites of theribotoxin molecule that may be modified according to the presentinvention without substantial loss of biological activity.

Intact eucaryotic ribosomes have a sedimentation coefficient of 80S andare composed of 60S and 40S subunits. The 60S subunit contains 28S rRNAabout 4700 nucleotides long. Alpha-sarcin produces a single fragment of393 nucleotides derived from the 3'end of 28S rRNA and the cleavageproduces 3'phosphate and 5'hydroxyl groups. The substrate must not befree rRNA since with free 28S rRNA the toxin causes extensivedegradation of the nucleic acid. The fragment can also be generated fromintact 80S ribosomes but not from the 40S ribosomal subunit. Due to theextremely selective action of this class of ribotoxins the structure hasbeen highly conserved. The ability to cleave a single phosphodiesterbond of 28S rRNA in a 60S ribosomal subunit may be assayed according tothe method of Endo, Y. & Wool, I. G. (1982), J. Biol. Chem. 257,9054-9060. Preferably the protein analogue has at least 5% of thepotency of the native sequence ribotoxin, more preferably the proteinanalogue has at least 10% of the potency of the native sequenceribotoxin and especially the protein analogue has at least 12% of thepotency of the native sequence ribotoxin. Potency is determined byinhibition of protein synthesis according to section 8 below.

The term "one cysteine available for covalent linkage" as used hereinmeans that whilst the protein analogue may or may not contain othercysteines only one is readily capable of forming the covalent linkage toa partner using conjugation techniques known in the art. Usually thenative ribotoxin contains other cysteines not readily available forcovalent linkage to a partner. Cysteines not available for covalentlinkage to a partner may for example be part of disulphide bridges orburied within the centre of the protein or within deep surface clefts.Preferably the one cysteine available for covalent linkage is present onthe surface of the protein and preferably positioned away from anyregion associated with biological activity of the protein such as forexample the active site.

"Partner" refers to a molecule which may be an individual molecule orpart of a macromolecule, polymer or solid phase. Preferably the partneris an agent that selectively binds to tumours such as for exampleimmunoglobulin or interleukin 2, more preferably a protein whichselectively binds to tumours, more preferably an immunoglobulin whichselectively binds to tumours and especially an immunoglobulin class Gwhich selectively binds to tumours. A preferred antibody for selectivelybinding to colorectal rumours is monoclonal antibody C242 as describedin international patent application WO 92/01470. "Immunoglobulin" and"antibody" refer to whole molecules as well as fragments thereof, forexample known fragments such as for example Fab, Fv and single chain Fv(Methods in Enzymology 178, Academic Press 1989 and Bio/Technology 9,545-551, 1991).

The term "native ribotoxin" includes ribotoxins as found in nature orrecombinant versions thereof and analogues of such ribotoxins which haveno cysteines available for covalent linkage to a partner or more thanone cysteine available for covalent linkage to a partner. Preferablenative ribotoxins are restrictocin, mitogillin and alpha-sarcin, morepreferably restrictocin and mitogillin and especially restrictocin. Theprotein analogue may be derived from the native ribotoxin or fromanother analogue of the native ribotoxin by recombinant DNA techniquessuch as for example by site directed mutagenesis. For example if thenative ribotoxin comprises more than one cysteine available for covalentlinkage then the derivation of the analogue will involve removal ofcysteines until only one remains. Preferably on the other hand if thenative ribotoxin comprises no cysteines available for covalent linkagethen the derivation of the analogue will involve addition of a cysteine.Removal or addition of a cysteine may involve substitution of anexisting amino acid of the native ribotoxin. Thus a cysteine availablefor covalent linkage may be added by substitution of one cysteine in anon-essential disulphide bridge leaving the other cysteine available forcovalent linkage to a partner. In general however disulphide bridges donot represent a preferred substitution site. A cysteine may also beadded by an extension to the N or C terminus of the native ribotoxin orfrom another analogue of the native ribotoxin preferably of 1-50 aminoacids, more preferably 1-40 amino acids, more preferably 1-30 aminoacids, more preferably 1-20 amino acids, more preferably 1-10 aminoacids, more preferably 1-5 amino acids and especially 2 amino acids. Theprotein analogue may also be prepared via total gene synthesis fromoligonucleotides. Oligonucleotide synthesis has been reviewed by M. J.Gait in Oligonucleotide Synthesis, IRL Press 1984. Total gene synthesishas been described by H. Edwards in International Biotechnology Lab.5(3) 19-25, 1987.

Preferred restrictocin analogues comprise:

i) restrictocin analogues in which any one of the following residues innative restrictocin comprises cysteine: Lys 13, Lys 20, Lys 28, Lys 60,Lys 63, Lys 69, Ser 82, Lys 88, Lys 106, Lys 110 and Lys 128; and

ii) native restrictocin comprising the C-terminus extension Cys 150-Gly151; and

iii) native restrictocin comprising the N-terminus extension Gly-Cysrestrictocin.

Especially preferred restrictocin analogues comprise:

i) restrictocin analogues in which any one of the following residues innative restrictocin comprises cysteine: Lys 13, Ser 82, Lys 106 and Lys110; and

ii) native restrictocin comprising the C-terminus extension Cys 150-Gly151.

Preferred mitogillin analogues comprise:

i) mitogillin analogues in which any one of the following residues innative mitogillin comprises cysteine: Lys 13, Lys 20, Lys 28, Lys 60,Lys 63, Lys 69, Ser 82, Lys 88, Lys 106, Lys 110 and Lys 128; and

ii) native mitogillin comprising the C-terminus extension Cys 150-Gly151; and

iii) native mitogillin comprising the N-terminus extension Gly-Cysmitogillin.

Especially preferred mitogillin analogues comprise:

i) mitogillin analogues in which any one of the following residues innative mitogillin comprises cysteine: Lys 13, Ser 82, Lys 106 and Lys110; and

ii) native mitogillin comprising the C-terminus extension Cys 150-Gly151.

Preferred alpha-sarcin analogues comprise:

i) alpha-sarcin analogues in which any one of the following residues innative alpha-sarcin comprises cysteine: Lys 14, Lys 21, Lys 29, Lys 61,Lys 64, Lys 70, Ser 83, Lys 89, Lys 107, Lys 111 and Lys 129; and

ii) native alpha-sarcin comprising the C-terminus extension Cys 151-Gly152; and

iii) native alpha-sarcin comprising the N-terminus extension Gly-Cysalpha-sarcin.

Especially preferred alpha-sarcin analogues comprise:

i) alpha-sarcin analogues in which any one of the following residues innative alpha-sarcin comprises cysteine: Lys 14, Ser 83, Lys 107 and Lys111; and

ii) native alpha-sarcin comprising the C-terminus extension Cys 151-Gly152.

According to another aspect of the present invention there is provided aconjugate comprising a partner covalently linked to a protein analogueas described above. Preferably the conjugate is an immunotoxin andespecially a tumour selective immunotoxin. Preferably the conjugate hasa potency, as determined by the in vitro cytotoxicity assay set out insection 9.1, of 10E-7M or less, more preferably the conjugate has apotency of 10E-8M or less and especially the conjugate has a potency of10E-9M or less.

According to another aspect of the present invention there is provided apolynucleotide sequence capable of encoding a protein analogue asdescribed above. The term "capable of encoding" refers to the degeneracyof the genetic code wherein some amino acids are encoded by more thanone triplet of nucleotides or codon.

According to another aspect of the present invention there is provided areplicative cloning vector comprising the polynucleotide sequencedescribed above such as for example a plasmid.

According to another aspect of the present invention there is provided areplicative expression vector comprising the polynucleotide sequencedescribed above such as for example a plasmid.

According to another aspect of the present invention there is providedrecombinant host cells transformed with a replicative expression vectoras described above. The host cells may be procaryotic or eucaryotic suchas for example bacterial, yeast or mammalian cells.

According to another aspect of the present invention there is provided amethod of producing a protein analogue which comprises culture of therecombinant host cells described above. Recombinant restrictocinexpressed in E. coli may be produced in either soluble form(particularly by manipulation of expression construct and growthconditions) or as insoluble inclusion bodies. Expression of eucaryoticpolypeptides in E.coli is known in the art and has been reviewed byMarston, F. A. O. in Biochem. J. 1986, 240, 1-12 and in Methods inEnzymology 185, Academic Press 1990. Purification of proteinsaccumulating in soluble form may be accomplished by cell lysis(sonication or high pressure homogenisation), centrifugation,ion-exchange chromatography, gel permeation chromatography, ammoniumsulphate precipitation, acid precipitation, triazine dye chromatography,in a variety of combinations. Renaturation and purification of insolublerestrictocin may be accomplished by cell lysis (sonication or highpressure homogenisation), centrifugation, washing of the pellet fractionwith buffer or mild chaotropes, solubilisation in chaotropic agents (eg.6M guanidine HCl, 8M urea, detergents etc with or without reductantbeing present) followed by the controlled removal of the chaotropicagent (eg. by dilution or dialysis) and formation of the disulphidebonds by oxidation reactions (eg. using dissolved air, glutathione redoxbuffers, etc). The resulting solubilised, renatured restrictocin maythen be purified.

Alternatively restrictocin may be expressed by suitable yeast expressionsystems with secretion of the mature protein into the culture supernateusing known techniques (Methods in Enzymology 194, Academic Press 1991and Methods in Yeast Genetics by Rose, M. Winston, F. & Hieter, P., ColdSpring Harbour Press 1990). The expression system may incorporate yeastsecretion sequences or Aspergillus secretion sequences such as forexample restrictocin presequences to facilitate secretion of the activeprotein into the medium in an effective manner. Purification from yeastsupernates may be achieved using standard methods.

According to another aspect of the present invention there is provided apharmaceutical composition comprising a protein analogue covalentlylinked to a partner as an active ingredient in association with apharmaceutical excipient or carrier. The composition will convenientlycomprise an effective amount of the said active ingredient.

According to another aspect of the present invention there is provided apharmaceutical composition comprising a conjugate as described above asan active ingredient in association with a pharmaceutical excipient orcarrier. Preferably the conjugate is an immunotoxin and especially atumour selective immunotoxin. The composition will conveniently comprisean effective amount of the said active ingredient.

Pharmaceutical compositions of the present invention may be in a varietyof dosage forms. Generally, the pharmaceutical compositions of thepresent invention will be administered parenterally, preferablyintravenously. A particular parenteral pharmaceutical composition is onewhich is formulated in a unit dosage form which is suitable foradministration by injection. Thus, particularly suitable compositionscomprise a solution, emulsion or suspension of the immunoconjugate orprotein analogue linked to a partner in association with apharmaceutically acceptable parenteral carrier or diluent. Suitablecarriers or diluents include aqueous vehicles, for example water orsaline, and non-aqueous vehicles, for example fixed oils or liposomes.The compositions may include agents which enhance stability. Forexample, the composition may include a buffer. The concentration of theactive ingredient will vary, but in general, the active ingredient maybe formulated at concentrations of about 1 to 10 mg/kg.

According to another aspect of the present invention there is provided amethod of treatment of a tumour or tumours comprising the administrationto a human or animal in need of such treatment of a pharmaceuticallyeffective amount of a conjugate as described above. Preferably theconjugate is an immunotoxin and especially a tumour selectiveimmunotoxin.

According to another aspect of the present invention there is provided aconjugate as described above for use as an active therapeutic substance.Preferably the conjugate is an immunotoxin and especially a tumourselective immunotoxin.

According to another aspect of the present invention there is providedthe use of a protein analogue to prepare a conjugate such as for examplean immunotoxin.

According to another aspect of the present invention there is provided amethod of preparing a conjugate which method comprises linkage of aprotein analogue as described above to a partner.

The invention will now be illustrated but not limited by reference tothe following examples.

EXAMPLE 1 Protein Analogue Structure

The structures of restrictocin (SEQ ID NO:10), mitogillin (SEQ ID NO:47)and alpha-sarcin (SEQ ID NO:48) have been compared by Lamy, B. & Davies,J. in Nucleic Acids Research, 1991, 19, 1001-1006. Restrictocin is a 149amino acid protein produced by Aspergillus restrictus. It contains 4cysteine residues thought to form 2 disulphide bonds. Mitogillin isidentical to restrictocin except for position 52 where serine inrestrictocin is replaced by asparagine in mitogillin. Restrictocin andalpha-sarcin are about 86% homologous in their amino acid sequences. Dueto the extremely selective action of this class of proteins thestructure has been highly conserved.

Protein analogues of the present invention include restrictocin andmitogillin in which a C-terminal extension Cys 150-Gly 151 is added andalpha-sarcin in which a C-terminal extension Cys 151-Gly 152 is added.Protein analogues of the present invention include restrictocin andmitogillin in which Lys 13 is replaced with cysteine and alpha-sarcin inwhich Lys 14 is replaced with cysteine.

Protein analogues of the present invention include restrictocin andmitogillin in which Lys 106 is replaced with cysteine and alpha-sarcinin which Lys 107 is replaced with cysteine.

Protein analogues of the present invention include restrictocin andmitogillin in which Lys 110 is replaced with cysteine and alpha-sarcinin which Lys 111 is replaced with cysteine.

Protein analogues of the present invention include restrictocin andmitogillin in which Ser 82 is replaced with cysteine and alpha-sarcin inwhich Ser 83 is replaced with cysteine.

Regions of the proteins generally unsuitable for preparation ofanalogues of the present invention are outlined below. The loop formedby residues 34-43 is close to the active site region of restrictocin andhence changing Lys 42 to cysteine could result in a protein withcompromised activity. Similar considerations apply to the correspondingresidues in alpha-sarcin.

In addition changing the potential `catalytic` residues His 136 and Glu95 of restrictocin may result in an inactive protein. Substitution withcysteine residues adjacent to the existing cysteine residues in themolecule could result in the formation of incorrect disulphide bridgeswhich, again, could lead to the formation of inactive molecularstructures. Similar considerations apply to the corresponding residuesin alpha-sarcin.

Substitution of residues which may be important in maintaining theoverall conformation of the structure (in addition to the cysteineadjacent positions described above) could also lead to inactive proteindue to deformation of the active site region or more grossconformational changes or structures which are unable to fold into anactive structure. Such residues in restrictocin could include prolineresidues (eg. Proline 97, Pro 100, Pro 112, Pro 118, Pro 126 etc) orresidues in the single alpha helix structure in the molecule (residues15-28). Similar considerations apply to the corresponding residues inalpha-sarcin.

EXAMPLE 2 Preparation of Genes Encoding Restrictocin Analogues

At all stages of the methodology, except where stated otherwise,standard molecular biological techniques were performed according toManiatis, T. et al. (1989) Molecular Cloning--A Laboratory Manual, ColdSpring Harbour Press, New York.

2.1 Cloning of a Restrictocin Coding Sequence from Aspergillusrestrictus

Aspergillus restrictus was obtained from the American Type CultureCollection (ATCC reference 34475). Spores of the fungus were used toinoculate medium containing 2% w/v soybean meal, 2% w/v corn meal, 1%w/v corn steep liquor, 0.5% w/v calcium carbonate, 1% w/v peptone and0.5% v/v antifoaming agent comprising 3% w/v octadecanol in lard oil. 50ml cultures were grown in 250 ml conical flasks, shaken at 30° C. for 48hours.

Genomic DNA was isolated from Aspergillus restrictus as described below.Nine shake flask cultures as above were filtered through a 0.2micrometer filter. Approximately 5 centimeter cubed portions of thefilter retained material were spooned into liquid nitrogen and blended(10 fifteen second pulses) on the "high" setting in a Waring blender.The blended material was transferred to a beaker and the remainingliquid nitrogen allowed to evaporate. To this mixture was added 150 ml0.3M sodium acetate, 15 ml 20% w/v sodium dodecyl sulphate and 15 mlphenol/chloroform (prepared as described in Maniatis et al, MolecularCloning; A Laboratory Manual, Cold Spring Harbor Laboratory Presspp.458-459, 1982). The mixture was stirred for 5 minutes. The resultingemulsion was centrifuged at 20000 g for 5 minutes. DNA was precipitatedfrom the aqueous phase by addition of ethanol (450 ml) andcentrifugation at 20000 g for 5 minutes. The pellet was rinsed with 70%v/v ethanol (50 ml), dried in vacuo and resuspended in TE buffer pH8.0(2 ml, prepared as described in Maniatis et al, Molecular Cloning; ALaboratory Manual, Cold Spring Harbor Laboratory Press p448, 1982)containing ribonuclease A (25 microliters of 100 micrograms per ml in TEas above, heated at 100° C. for 5 minutes). TE buffer consists of 10 mMTris-HCl, 1 mM EDTA at pH 8. The resulting solution was incubated at 37°C. for 150 minutes then water (7 ml) and 3.0M sodium acetate (1 ml) wasadded. The DNA was precipitated by the addition of ethanol (30 ml) andcentrifugation at 12000 g for 1 minute. The pellet was resuspended in0.3M sodium acetate (10 ml) and extracted with phenol/chloroform (5 ml,as above). The resulting emulsion was centrifuged at 12000 g for 5minutes. DNA was precipitated from the aqueous phase by addition ofethanol (30 ml) and centrifugation at 12000 g for 1 minute. The pelletwas rinsed with 70% v/v ethanol (40 ml), dried in vacuo and resuspendedin TE buffer pH8.0 (5 ml, prepared as described above). This Aspergillusrestrictus genomic DNA solution was stored at -20° C.

A portion of the above generated DNA containing the coding sequence formature restrictocin was amplified using the polymerase chain reaction asdescribed by Kleppe et al in J. Mol. Biol., 56, 341-361, (1971), andSaiki et al in Science, 239, 487-491, (1988). The polymerase chainreaction was performed using the thermostable DNA polymerase isolatedfrom the bacterium Thermus aquaticus described by Chien et al inBiochemistry, 27, 1550-1557, (1976). Oligodeoxyribonucleotide(hereinafter referred to as oligonucleotide) primers were designed forthe polymerase chain reaction according to the possible DNA codingsequences for restrictocin. This was done using the primary (amino acidsequence) structure of restrictocin reported by Lopez-Otin et al (Eur.J. Biochem. 143, p621-634). The nucleotide sequences of theoligonucleotide primers were variable at specific positions to allow forthe degeneracy of the genetic code. Special features were introducedinto the oligonucleotide primer sequences however such that not allpossible codons for a given amino acid were present. The rationale beingto maximise codon usage to those codons found in highly expressed genesof E. coli. This is thought to be particularly important at the 5' endof the coding sequence. A further feature of the design of theoligonucleotide primers (not subsequently used) was to introduce theability of the primers to generate analogues of the protein encoded bythe DNA sequence bounded by the oligonucleotide primers when used in thepolymerase chain reaction. A further feature of oligonucleotide primerdesign was the introduction of restriction enzyme recognition sequencesto facilitate the cloning of polymerase chain reaction products derivedfrom the use of the oligonucleotide primers.

Polymerase chain reaction amplification of the Aspergillus restrictusgenomic DNA was effected by combining 1 microliter of the solution with100 picomoles oligonucleotide primer SEQ.ID NO. 1, 116 picomolesoligonucleotide primer SEQ ID. NO. 2 and 1.25 units Thermus aquaticusDNA polymerase (Cetus "Amplitaq") in a 100 microliter solution that alsocontained (final concentrations) 100 micromolar each of the fourdeoxynucleoside triphosphates, dATP, dTTP, dCTP and dGTP, 1.2 mMmagnesium chloride, 10 mMTris/HCl pH8.3, 50 mM potassium chloride and0.01% w/v gelatin. This solution was overlaid with light mineral oil(Sigma) and subjected to thermal cycling. The thermal cycling comprised10 cycles of 94° C. for 1 minute, 37° C. for 2 minutes and 55° C. for 2minutes then 20 cycles of 94° C. for 1 minute, 60° C. for 2 minutes and72° C. for 2 minutes and the final 72° C. incubation was extended to 5minutes. After gel electrophoresis the main product was isolated usingDEAE cellulose membrane (NA45 paper) as described in Maniatis, T. et al.(1989) Molecular Cloning--A Laboratory Manual, Cold Spring HarbourPress, New York. The method described in Maniatis is based on that ofDretzen et al. (1981) Anal. Biochem. 112, 295. NA45 paper is a DEAEcellulose membrane supplied by Schleicher and Schull.

The initial intention was to clone the PCR product into M13mp11 viaPvuII and Sal I sites as the oligonucleotides SEQ. ID NOS. 1 and 2contained these respective recognition sequences. However, sequencing ofsuch M13 clones showed the restrictocin gene to be truncated, which wassuspected and later confirmed to be due to a PvuII recognition sequencewithin the restrictocin gene sequence. Therefore, a second PCR reactionwas performed to introduce other cloning sites into the PCR product, 5'to the restrictocin gene. Approximately 1 microliter of eluate wasreamplified, as above, with 100 picomoles each of oligonucleotideprimers SEQ ID NOS. 3 and 4. The thermal cycling comprised 5 cycles of94° C. for 1 minute, 37° C. for 2 minutes and 55° C. for 2 minutes then25 cycles of 94° C. for 1 minute, 60° C. for 2 minutes and 72° C. for 2minutes and the final 72° C. incubation was extended to 5 minutes. Themain product was isolated after agarose gel electrophoresis using NA45paper as described above.

This purified PCR product was digested with BamHI and SalI and ligatedinto SalI and BamHI cleaved M13mp11 using T4 DNA ligase. Ligation mixeswere used to transfect E.coli, strain TG1. TG1 was supplied with theOligonucleotide-Directed In-Vitro Mutagenesis System Version 2 suppliedby Amersham (code RPN-1523) and was suitable for the site directedmutagenesis protocol described below but other M13 host strains whichcarry the F-pilus may also be used such as for example JM101 (ATCC No.33876). Strain TG1 has a published genotype K12, delta(lac-pro),supE,thi,hsdD5/F'traD36, proA⁺ B⁺, lacI_(q), lacZ delta M15 (Gibson1984, Ph.D. Thesis, University of Cambridge, U.K.) and TG1 is freelyavailable from for example the E. coli Genetic Stock Centre, YaleUniversity, USA. Restrictocin sequences were checked using the dideoxychain termination approach such as for example using the Sequenaseversion 2 sequencing kit supplied by United States Biochemicals.Initially M13 and M 13 reverse sequencing primers were used (SEQ. IDnos.5 and 6). The restrictocin sequence was completed using sequencingprimers with SEQ ID NOS. 7, 8 and 9. A sequence (SEQ. ID. NO. 10) codingfor mature, wild-type restrictocin was finally obtained. The flankingsequences up to the BamHI and SalI cloning sites were as follows:

At the 5' end of the restrictocin coding sequence: ##STR1## At the 3'end of the restrictocin coding sequence: ##STR2##

In order to make subsequent subcloning manipulation easier theM13-restrictocin clone was digested with restriction endonucleases SalIand HindIII. The small (approx 0.6 Kb) SalI-HindIII fragment from pBR322was then cloned into the M13-restrictocin clone backbone. This had theeffect of deleting the PstI recognition site adjacent to the SalI sitesituated 3' of the restrictocin gene.

The restrictocin coding sequence obtained was consistent with thepublished amino acid sequence for mature restrictocin except at position115. The published amino acid sequence disclosed an asparagine residueat position 115 (Lopez-Otin et al (1984), 143, p621-634), but oursequenced PCR products indicated there was an aspartic acid residue(GAC) at position 115. The publication of the genomic sequence forrestrictocin (Lamy, B. and Davies, J. (1991) Nucleic acids Res. 19(5) ,p1001-1006) supported our assignment suggesting the originally publishedamino acid sequence was incorrect. Hence, our restrictocin codingsequence (sequence ID no. 10) codes for the same protein as predictedfor mature restrictocin from the genomic sequence. However, it should beemphasised that the nucleotide sequence was different at the 5' (and 3')ends due to the use of PCR to clone the gene.

2.2 Generation of Coding Sequences for Restrictocin Analogues

The purpose of cloning the restrictocin coding sequence into an M13vector was to allow site-directed mutagenesis and so generate analogueswith desired characteristic(s). Mutagenesis was performed using theOligonucleotide-Directed In-Vitro Mutagenesis System Version 2 suppliedby Amersham (code RPN-1523). This kit was based on the method of Sayerset al ((1988) Nucleic Acids Res., 16, p791-802). A 5'phosphorylatedmutagenic oligonucleotide (see below) was annealed to thesingle-stranded template (the M13 restrictocin plasmid described above)and extended by Klenow polymerase in the presence of T4 DNA ligase togenerate a mutant heteroduplex. Selective removal of the non-mutantstrand was made possible by the incorporation of a thionucleotide intothe mutant strand during the above in vitro synthesis. Cutting with arestriction endonuclease (NciI), which cannot cleave phosphorothioateDNA, resulted in generation of a nick in the template strand only. Thisnick presented a site for exonuclease III which was used to digest awaypart of the non-mutant (non-phosphorothioate) strand of the restrictocinsequence. The mutant strand was then used as a template to reconstructthe double-stranded closed circular molecule, by DNA polymerase I and T4DNA ligase treatment, creating a homoduplex molecule. The sequence forrestrictocin analogues TR3/TR4/TR5/TR6/TR1 which contained a Ser82 toCys82 substitution/a Lys106 to Cys106 substitution/a Lys13 to Cys13substitution/a C-terminal extension of Cys150-Gly151/a Lys 110 to Cys110 substitution respectively was generated using 5'phosphorylatedmutagenic oligonucleotides with SEQ ID NO. 13: CCGAAGCACT GCCAGAACGGC/SEQ ID NO 14: CACGACTATT GCTTTGACTC G/SEQ ID NO. 15: CTGAATCCCTGCACAAACAA A/SEQ ID NO. 16: CTGTGTAGCC ACTGCGGTTA ATAATAGTCG/SEQ ID NO.41: TTTGACTCGT GCAAACCCAA G respectively.

EXAMPLE 3 Expression of Restrictocin and Analogues

3.1 Generation of the Restrictocin Expression Vector

The generation of a restrictocin expression vector containing thelambdaPL promoter was initiated with the precursor plasmids, pICI0074and pICI1079. Vector pICI1079 has been deposited under the BudapestTreaty at the National Collections of Industrial and Marine BacteriaLimited (NCIMB), 23 St. Machar Drive, Aberdeen, AB2 1RY, Scotland, U.K.(NCIMB No. 40370), date of deposit 19 Feb. 1991). The production ofpICI0074 is described in European Patent Publication No 459630A2published Dec. 4, 1991 and pICI1079 is also described therein.

pICI0074 and pICI1079 were digested with EcoRI and SacI. The fragmentswere then put into a ligation reaction, and the ligation reactionmixture used for transformation of E. coli. Screening by restrictionmapping was used to identify the recombinant plasmid in which thelambdaPL/CI857 repressor fragment from pICI1079 is inserted into thepICI0074 backbone fragment which contains the tetracycline genes, a cerstability function, multiple restriction cloning sites and the T4transcription terminator. A linker sequence containing a ribosomebinding site sequence was cloned between the SacI and KpnI sites (ie.into the polylinker) of the above generated intermediate vector. Thelinker for generation of expression vector pICI 0122 (lambdaPL-RBS7) wasmade through hybridisation of the two 5' phosphorylatedoligonucleotides, SEQ. ID. NOS. 11 and 12: ##STR3##

The restrictocin coding sequence (SEQ. ID. No. 10) and the analogues ofrestrictocin described above were subcloned from the M13 restrictocinclones into lambdaPL-RBS7 using the subcloning strategy now detailed togenerate restrictocin expression vectors. The vector encoding wild typerestrictocin was called pICI 1453 (lambdaPL-RBS7-RES), the vectorencoding the analogue with Ser82 to Cys82 substitution was called pICI1485, the vector encoding a Lys106 to Cys106 substitution was calledpICI 1486, the vector encoding a Lys13 to Cys13 substitution was calledpICI 1487, the vector encoding the C-terminal extension of Cys150-Gly151was called pICI 1488 and the vector encoding Lys 110 to Cys 110substitution was called pICI 1472.

Initially, pICI 0122 was digested with KpnI and the overhang blunt-endedusing T4 DNA polymerase. It was then further digested with XhoI (likeKpnI, situated in the polylinker) before treating with calf intestinalalkaline phosphatase to prevent subsequent religation of the fragments.The M13-restrictocin clones containing either the wild type restrictocinor restrictocin analogues were digested with PstI and then the overhangswere blunt-ended with T4 DNA polymerase. Digestion by SalI released therestrictocin coding sequence. A ligation reaction was then performed toinsert the restrictocin fragment into the pICI 0122 backbone. At the 5'end of the restrictocin sequence the ligation is blunt-ended, but theSalI overhang at the 3' end was compatible with the XhoI site. ThePstI/blunt ending reaction results in the 5' most base of therestrictocin fragment being the first base of the first codon of therestrictocin coding sequence. The KpnI/blunt ending reaction results inthe 3' end of the expression vector backbone reading ATG which was theinitiation codon corresponding to the RBS sequence directly upstream.Hence the ligations resulted in fusion of restrictocin coding sequencesin frame with the translation initiation codon.

3.2 Expression Studies

Initially, following characterisation, pICI1453 (lambdaPL-RBS7-RES) wastransformed into E.coli MSD 462. 75 ml of M9 medium supplemented with0.02% casein acid hydrolysate (Oxoid L41) and 15 μg/ml tetracycline wasinoculated with a single colony from a fresh plate and grown overnightat 35° C. with gentle shaking. M9 medium comprises 6 g/l di-sodiumhydrogen orthophosphate, 3 g/l potassium dihydrogen orthophosphate, 0.5g/l sodium chloride, 1.0 g/l ammonium chloride, 1 mM magnesium sulphate,0.1 mM calcium chloride, 2 g/l glucose and 4 μg/ml thiamine. The OD₅₅₀was measured and the culture diluted with the same medium to give a 75ml volume with OD₅₅₀ =0.1. This culture was grown at 37° C. with gentleshaking until OD₅₅₀ =0.4-0.6 (approx 3-4 hrs.). The incubatortemperature was increased to 42° C. and growth continued for a further 3hours, to allow induction of restrictocin expression. However, SDS-PAGEgels of whole cell lysates did not detect restrictocin expression.Subsequently, 35-S methionine pulse chase labelling was used todemonstrate that the short half life of restrictocin was drasticallylimiting its accumulation. We can overcome this instability problem byusing the protease deficient strain MSD460. The growth and inductionprotocol is as for MSD462 except the M9 medium is further supplementedwith 45 mg/l methionine. MSD460(pICI 1453) gives restrictocinaccumulation levels of 5-10% total cell protein and similar levels ofrestrictocin analogue accumulation are obtained with the restrictocinanalogue expression vectors pICI 1485, pICI 1486, pICI 1487, pICI 1488and pICI 1472.

Other promoter systems based on standard techniques may be used.

3.4 Derivation of MSD460 (Host Strain for Restrictocin Expression)

The lon allele was introduced into MSD 101 (E. coli W3110) by P1transduction from SG20252 (Trislar and Gottesman (1984) J. Bacteriol.160, 184-191) and selection for tetracycline resistance followed byscreening for sensitivity to nitrofurantoin. SG20252 had thetetracycline resistance transposon Tn10 closely linked to the lon100allele. Nitrofurantoin sensitivity was characteristic of ion-strains.One of the resultant clones was termed MSD310. Presence of thetransposon in this strain was undesirable and a derivative lacking thiselement was isolated by screening for spontaneously arising clones whichhad lost tetracycline resistance. One of these, termed MSD413 wasisolated and shown to retain nitrofurantoin sensitivity. This strain wasmucoid, characteristic of Lon⁻ strains. To eliminate this phenotypewhich was due to over production of capsular polysaccharide phage Mumutagenesis using a derivative of phage Mu termed Mu cts d1 ApR lac wasemployed. Following infection of MSD413 with Mu cts d1 ApR lac,ampicillin resistant clones were selected on L-amp plates and screenedfor a non-mucoid phenotype. One such clone termed MSD413*2 was isolatedand shown to retain nitrofurantoin sensitivity. We have called themutation in this strain which suppresses over production of capsularpolysaccharide som-6. Presence of Mu cts d1 ApR lac in this strain isundesirable. A derivative which has lost the defective phage, but hasretained som-6 was isolated by conventional heat curing and screeningfor loss of ampicillin resistance. One such clone, which appeared toretain som-6 and which was nitrofurantoin sensitive was termed MSD460.

On more extensive characterization, MSD460 was found to requiremethionine, but not histidine for growth (i.e was Met-) and was unableto grow on arabinose as sole source of carbon although it retained theability to grow on glycerol. The most likely interpretation of thesedata is that MSD460 carries a deletion extending through metG and araFGat minute 45 of the E. coli genetic map, but which does not extendbeyond the bis operon at minute 44 and the glp operon at minute 48.5.

MSD 460 has been deposited under the Budapest Treaty with the NationalCollection of Industrial and Marine Bacteria Limited (NCIMB), 23 StMacbar Drive, Aberdeen AB2 1RY, Scotland, United Kingdom. The accessionnumber is NCIMB 40469 and the date of deposit was 9-Jan.-92.

EXAMPLE 4 Generation and Expression of Alpha-Sarcin Analogues

4.1 Construction of a Synthetic Gene Encoding Alpha-Sarcin

The construction of a synthetic alpha-sarcin gene and its expression inE.coli has already been described (Henze et al. (1990) Eur. J. Biochem.,192, p127-131) (SEQ ID NO:48). However, following the successfulexpression of restrictocin intracellularly in E.coli, there appears noneed to follow the published strategy of using secretion vectors toovercome potential toxicity problems.

The alpha-sarcin synthetic gene (coding sequence) is constructed in anidentical fashion to that described in the Henze publication, except theoligonucleotide pairs used to construct the 5' and 3' ends of the geneare different and a base pair in one of the internal oligonucleotidepairs is different. Additional guidelines are given regarding syntheticgene synthesis in Edwards, M. (1987) International Biotechnology Lab5(3), 19-25.

The oligonucleotide pairs used to construct the 5' end of the gene haveSEQ ID nos. 26 and 27 (replacing oligonucleotides 1 and 2 in thepublication). This oligonucleotide pair generate a PstI overhang beyondthe coding sequence.

The oligonucleotide pairs used to construct the 3' end of the gene haveSEQ ID nos. 28 and 29 (replacing oligonucleotides 18 and 19 in thepublication). This oligonucleotide pair generates a Bam HI overhang andintroduces a SalI site internal to this overhang, downstream of thealpha-sarcin stop codon.

The oligonucleotides 6 and 7 in the publication are replaced with SEQ IDnos 37 and 38. This removes the BamHI site but does not change the aminoacid sequence.

The assembled gene is then cloned into the PstI and BamHI sites ofM13mp10 via the above mentioned PstI and BamHI overhangs.

4.2 Generation of Coding Sequence for an Alpha-Sarcin Analogue

Analogues of alpha-sarcin are generated using the same M13-site directedmutagenesis methodology as described for restrictocin. The analoguesdescribed herein and the 5' phosphorylated mutagenic oligonucleotidesused for their generation are as follows:

Lys 14 to Cys 14: seq ID no. 35

Ser 82 to Cys 82: seq ID no. 31

Lys 107 to Cys 107: seq ID no. 34

C-terminal extension of Cys 151--Gly 152: seq ID no. 36

Lys 111 to Cys 111: seq ID no. 42

4.3 Construction of Alpha-Sarcin Analogue Expression Vector andExpression

Following mutagenesis and characterisation, the alpha-sarcin analogue isthen cloned into the expression vector, pICI0122 (LambdaPL-RBS7) asdescribed for restrictocin and its analogues. In summary, theM13--alpha-sarcin analogue clone is digested with PstI, blunt-ended withT4 DNA polymerase, and then digested with SalI. pICI0122 is cut withKpnI, blunt-ended with T4 DNA polymerase and then digested with XhoI. Aligation reaction is then performed to introduce the alpha-sarcin codingsequence into the opened vector polylinker. This results in the ATGinitiation codon in the vector being fused blunt ended and in-frame withthe first codon of alpha-sarcin.

The alpha-sarcin expression vector is then intoduced into the proteasedefiecient strain MSD460 and induction of expression performed asdescribed above for restrictocin and analogues.

EXAMPLE 5 Growth of E.coli in Fermenters and Expression of Restrictocinand Restrictocin Analogues

E.coli strain MSD460 was transformed with plasmid pICI 1453 and theresultant recombinant purified and maintained in glycerol stocks at -80°C. An aliquot of the culture was removed and streaked onto agar platesof L-tetracycline to separate single colonies after overnight growth at37° C. A single colony of MSD460 pICI1453 was removed and resuspended ina 10 ml L-tetracycline broth and 100 μl immediately inoculated into eachof 10 250 ml Erlenmeyer flasks containing 75 ml of L-tetracycline broth.After growth for 16 h at 37° C. on a reciprocating shaker the contentsof the flasks were pooled and used to inoculate a single fermentercontaining the growth medium described below.

The fermentation was carried out at a temperature of 37° C. and pH wascontrolled at 6.7 by automatic addition of 6M sodium hydroxide solution.The dissolved oxygen tension (dOT) set-point was 50% air saturation andwas contolled by automatic adjustment of the fermenter stirrer speed.Air flow to the fermenter initially 20 L/min (corresponding to 1 volumevolume per minute (VVM)) was increased to 2.5 VVM when the fermenterstirrer speed reached approximately 80-90% of its maximum. A solution ofyeast extract was fed into the fermenter at a rate of 1.7 g/L/h from 4.5h post inoculation until the culture OD₅₅₀ reached 50 when it wasincreased to 3.4 g/L/h.

Between 7-8 h post fermenter inoculation, when the supply of carbonsource (glycerol) in the fermentation became exhausted leading to arapid rise in dOT from 50% air saturation, a feed containing glycerol(714 g/L) and ammonium sulphate (143 g/L) was pumped into the fermenterat a rate which restricted the bacterial oxygen uptake rate (OUR) toapproximately 80% of the maximum oxygen transfer rate (OTR) of thefermenter (under the conditions described). The culture was induced byraising the fermentation temperature from 37° C. to 42° C. when theculture reached an OD₅₅₀ =80. The fermentation was maintained at 42° C.for 5 h. The bacteria were harvested in 1 L centrifuge bottles in aSorvall RC-3B centrifuge (7000× g, 4° C., 30 min) and the cell pastestored frozen at -80° C.

An essentially identical protocol was followed for growth of E.colistrains expressing restrictocin analogues.

    ______________________________________                                        Growth medium        g/L (deionized water)                                    ______________________________________                                        Potassium dihydrogen orthophosphate                                                                3.0                                                      di-Sodium hydrogen orthophosphate                                                                  6.0                                                      Sodium chloride      0.5                                                      Casein hydrolysate (Oxoid L.41)                                                                    2.0                                                      Ammonium sulphate    10.0                                                     Glycerol             35.0                                                     Yeast extract (Difco)                                                                              20.0                                                     Magnesium sulphate 7-hydrate                                                                       0.5                                                      Calcium chloride 2-hydrate                                                                         0.03                                                     Thiamine             0.008                                                    Iron sulphate 7-hydrate/Citric acid                                                                0.04/0.02                                                Trace element solution (TES)*                                                                      (0.5 ml/L)                                               Tetracycline         (10 mg/L)                                                ______________________________________                                                             mg/10 ml                                                 *Trace element solution                                                                            (deionized water)                                        ______________________________________                                        AlCl.sub.3.6H.sub.2 O                                                                              2.0                                                      CoCl.sub.2.6H.sub.2 O                                                                              0.8                                                      KCr(SO.sub.4).sub.2.12H.sub.2 O                                                                    0.2                                                      CuCl.sub.2.2H.sub.2 O                                                                              0.2                                                      H.sub.3 BO.sub.3     0.1                                                      KI                   2.0                                                      MnSO.sub.4.H.sub.2 O 2.0                                                      NiSO.sub.4.6H.sub.2 O                                                                              0.09                                                     Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                      0.4                                                      ZnSO.sub.4.7H.sub.2 O                                                                              0.4                                                      ______________________________________                                    

EXAMPLE 6 Purification of Protein Analogues

E.coli paste was resuspended in lysis buffer (1 g of wet cell paste per10 ml buffer) using a homogeniser (Polytron). Lysis buffer comprises 50mM Tris (hydroxymethyl) aminomethane hydrochloride, 2 mM(ethylenedinitrilo)tetraacetic acid (abbreviated EDTA), 0.02% Sodiumazide, pH8.2. The resuspended cells at 4° C. were then lysed by highpressure homogenisation (4 passes through Manton Gaulin homogeniser) orsonication (5×45 sec bursts) and the resulting lysed cell suspensioncentrifuged at 25,000 g for 20 minutes.

The pH of the supernatant containing soluble toxin was adjusted to pH 7by the addition of dilute hydrochloric acid, loaded onto a carboxymethylion exchange column (such as carboxy methyl Sepharose fast flow fromPharmacia) pre-equilibrated in 50 mM Tris/HCl, 2 mM EDTA, 0.02% Sodiumazide, pH 7. The column was eluted with a linear gradient of 0-1M sodiumchloride in the same buffer used for equilibration of the column. Theelution position of the toxin was determined by analysing the columnfractions by SDS-PAGE and fractions containing the toxin were pooled.

Pooled fractions were buffer exchanged into 25 mM sodium phosphate pH7.5 by dialysis or diafiltration, toxin samples which gave a precipitateafter dialysis were centrifugation at 40,000 g for 20 minutes and thesupernatant collected The toxin solution was loaded onto a dye affinitycolumn (such as mimetic green A6XL from ACL, Cambridge,U.K.)pre-equilibrated in 25 mM Sodium phosphate pH 7.5, and the flow throughsolution collected.

The column was washed with 25 mM sodium phosphate pH 7.5 and the washsolution collected. Contaminating proteins bound via adsorption to thedye ligand, the bulk of the toxin remained unbound and eluted in theflow through and wash. The elution position of the toxin was determinedby analysis of the column fractions by SDS-PAGE and toxin containingfractions pooled.

The pooled toxin containing fractions were judged >95% pure by SDS-PAGE.A small amount of disulphide bonded toxin dimer of molecular weight 34kDa was observed in some of the purified analogues (TR15%, TR4<5%,TR5<5%, TR6 20%). The identity of the 34 KDa band was confirmed bySDS-PAGE electroblotting and N-terminal sequencing (Problott, AppliedBiosystems, USA) [Matsuidara, J.Biol. Chem 262: 10035-38].

To confirm the purity of the purified toxin aliquots of the pooledfractions were subjected to SDS-PAGE and electroblotted. The 17 kDa bandwas excised and subjected to N-terminus sequence analysis on an AppliedBiosystems 475 protein sequencer (Applied Biosystems, USA). TR1, TR3 andTR4 gave the N-terminal sequence of restrictocin with no otherdetectable sequences. TR5 and TR6 gave N-terminal restrictocin sequencealong with that of another unidentified protein. Searches of Genembl,Swissprot and NBRF sequence databases failed to find any significanthomologies to known proteins. This contaminant was shown to represent 10% and 15% of the total protein for TR5 and TR6 respectively, asdetermined by reverse phase HPLC monitored at 214 nm and comparison ofthe peak areas. The identities of the peaks was confirmed by N-terminalsequencing.

6.1 Detailed Experimental Protocol for Purification of Analogue TR4

200 g of analogue TR4 E. coli paste was resuspended in 2 L of lysisbuffer (1 g per 10 ml) and lysed by sonication as described in section6. 1.8 L of the lysis supernatant following centrifugation andadjustment to pH 7 as described in section 6 was loaded onto acarboxymethyl Sepharose fast flow column (Pharmacia), [column bed volume290 mls, dimensions 5 cm i.d.×15 cm] at a flow rate of 8 mls a minute.After washing the column with the equilibration buffer until theabsorbance as monitored at 280 nm returned to baseline the toxin waseluted with a linear gradient of 0-1M sodium chloride (total volume 500mls) at 8 mls a minute. The toxin containing fractions were identifiedby SDS-PAGE analysis of aliquots of the fractions.

The toxin containing fractions were pooled (total volume 90 mls) anddialysed (Spectrapor 3.5 KDa cut off membrane, Pierce, U.S.A.) against 5L of buffer as described in section 6. The dialysed toxin solution wasloaded onto a mimetic green A6XL column (ACL, Cambridge, U.K.) [columnbed volume 150 mls, dimensions 2.6 cm i.d.×28 cm] at a flow rate of 4.25mls a minute and the flow through and wash collected as described insection 6. The toxin containing fractions were identified by SDS-PAGEanalysis of the fractions and pooled.

Samples of the pooled purified toxin were analysed by SDS-PAGE, SDS-PAGEelectroblotting and N-terminal sequencing, amino acid analysis and forbiological activity in the protein synthesis inhibition assay asdescribed in section 8.

The purified toxin was judged >95% pure by SDS-PAGE, with <5% disulphidebonded dimer, and the 17kDa SDS-PAGE blotted band gave only theN-terminal sequence of restrictocin. The yield was 7.2 mg of toxin asmeasured by amino acid analysis. The specific biological activity of thetoxin is shown in section 8.3.

The concentration of the final purified restrictocin analogues wasdetermined by amino acid analysis. The purified analogues wereconcentrated using membrane filtration before conjugation to antibody.

Samples of the purified analogues were assayed for protein synthesisinhibition activity as documented in section 8 below.

Toxin analogues TR1, TR3, TR5 and TR6 were purified and analysed usingthe procedures described for analogue TR4.

6.2 Purification of Recombinant Restrictocin

Recombinant restrictocin was purified as described for analogue TR4except that the pH of the lysis supernatant was adjusted to 8 prior toloading on the carboxymethyl Sepharose fast flow column, the column wasequilibrated and washed in pH 8 buffer, and the toxin eluted with alinear gradient of 0-0.5M sodium chloride in the same buffer. Also anadditional column step was used after the dye affinity column, thepooled toxin containing fractions were concentrated by ultrafiltration(Amicon stirred cell YM2 membrane) and chromatographed on a SephacrylS-200 HR size exclusion column. The column was pre-equilibrated with 20mM sodium phosphate, 150 mM sodium chloride, pH 7.5 and the sampleeluted with the same buffer. The elution position of the toxin wasdetermined by SDS-PAGE analysis of column fractions.

The purified restrictocin eluted with and apparent molecular weight ofabout 17 kDa, appeared >98% pure by SDS-PAGE and the SDS-PAGE blottedband gave the N-terminal sequence of restrictocin with no otherdetectable sequence.

EXAMPLE 7 Conjugation of Toxin Analogues to Antibody

Monoclonal antibody, such as for example the 19.9 antibody obtained fromhybridoma 1116NS-19 (ATCC No. HB 8059), in PBS at 12 mg/ml was dilutedwith 0.5 vols of borate buffer (100 mM sodium borate, pH 9.1). Theprotein concentration was determined by monitoring absorbance at 280 nmand the pH of the mixed solution noted (pH=8.7 to 8.9, preferably pH8.8).

Linker (N-succinimidyl 3-(2-pyridyldithio) butyrate) was dissolved indry, redistilled dimethylformamide or acetonitrile at a concentration of10 mg/ml and added immediately to the antibody solution, with mixing, ata ratio of 8 moles linker/mole antibody. The resulting solution wasincubated at 20° C. for one hour and applied to a size exclusiondesalting column equilibrated with 50 mM sodium phosphate/150 mM sodiumchloride/1 mM EDTA pH 8.0 to remove excess reagents and buffer exchangethe derivatised antibody. The desalted derivatised antibody was pooledand protein concentration determined by monitoring the absorbance at 280nm. The extent of derivatisation with linker was determined by additionof excess dithiothreitol to a sample of the derivatised antibody andmonitoring release of free thiopyridyl groups spectroscopically at 343nm. The extent of derivatisation was in the range 3 to 6 moles(preferably about 4 moles) linker groups per mole of antibody.

Recombinant toxin analogue was conjugated to derivatised antibody bymixing recombinant toxin analogue solution (in PBS adjusted to pH 8.0with sodium hydroxide) and derivatised antibody solution at a 1:1 w/wtoxin analogue/derivatised antibody ratio. The vessel was purged withargon and incubated at 15° C. for 40 h.

The protein solution was diluted with an equal volume of PBS adjusted topH 8.0 with sodium hydroxide and cysteine added to a final concentrationsufficient to cap excess linker groups on the antibody without reducingthe disulphide bond linking the toxin to the derivatised antibody.Completion of the cysteine capping reaction was confirmed by treating asample of the capped immunoconjugate with excess dithiothreitol andmonitored spectrophtometrically at 343 nm. No release of thiopyridylgroups was detected.

The immunoconjugate solution was concentrated by membrane filtration andapplied to a size exclusion chromatographic column equilibrated with 50mM sodium phosphate/25 mM sodium chloride/1 mM EDTA, pH6.3 and having afractionation range of 10-200 kd. The peak of protein containingimmunoconjugate was pooled and the protein concentration monitored byabsorbance at 280 nm.

EXAMPLE 8 Biological Assay for Ribotoxin Analogues

The aim was to establish conditions under which samples could be testedfor biological activity in a cell-free in vitro protein synthesis assay.

Rabbit reticulocyte lysates were prepared according to the method ofAllen and Schweet (J Biol Chem (1962), 237, 760-767). The assaydemonstrates inhibition of protein synthesis in a cell-free system by alack of incorporation of ¹⁴ C-labelled leucine into newly synthesisedprotein.

8.1 The Assay Protocol

Stock solution: 1 mM amino acid mix minus leucine. A solution containingall L-amino acids at 1 mM except leucine (adjusted to pH7.4 with NaOHand stored at -70° C.).

Soln. A

40 mM Magnesium acetate

2M Ammonium acetate

0.2M Tris

(pH 7.4 with HCl, stored 4° C.)

Soln. B

adenosine triphosphate (Sigma A5394) 246 mg/ml

guanosine triphosphate (Sigma G8752) 24.4 mg/ml

Assay mix:

1 ml Amino acid mixture

1 ml Soln. A

0.1 ml Soln. B

103 mg Creatine phosphate

1 mg Creatine kinase

510 μl H₂ O

600 μl (60 μCi) L-¹⁴ C-leucine (New England Nuclear, NEC-279E)

Reaction mix:

Test sample 25 μl

Assay mix 12.5 μl

Rabbit reticulocyte lysate 25 μl

Blank solution is 2 mg/ml bovine serum albumin(BSA) in phosphatebuffered saline(PBS)

All assays were performed in duplicate

12.5 μl of assay mix placed in sterile glass tubes

25 μl of BSA in PBS added to each of first four tubes for blanks

25 μl of test samples added to rest of tubes

1 ml 0.1M potassium hydroxide added to first two tubes (backgroundblank)

Tubes equilibrated to 28° C. in a water bath

25 μl of rabbit reticulocyte lysate (allowed to thaw from liquidnitrogen temperature) were added to each tube at 20 second intervals.When first tube had incubated for 12 minutes, 1 ml 0.1M KOH was added toeach tube again at 20 second intervals to allow all tubes to have 12minutes incubation. Two drops of 20% hydrogen peroxide were added toeach tube followed by 1 ml of 20% trichloroacetic acid (TCA). Tubes weremixed and allowed to stand for at least 1 hour, or overnight, at 4° C.The precipitates were filtered on to 2.5 cm glass fibre circle (GFC)discs, washed with 3×4 ml of 5% TCA, transferred to scintillation vialsand 10 ml scintillant (Ready-Solv. MP, Beckman) added. After 1 hour thevials were shaken and counted.

8.2 Establishment of Technique for use with E.coli Lysates

10 ml L-broth overnight cultures are grown at 37° C. 400 μl aliquots arepelleted at 13000 rpm for 30 seconds and most of the supernate decanted.

The pellets are subjected to 2 rounds of rapid freezing in solid carbondioxide/ethanol followed by thawing at 37° C. 12 μl of 25% sucrose in 50mM Tris HCl pH8.0 is added followed by 4 μl of a 10 mg/ml solution oflysozyme.

After incubation on ice for 15 minutes, 8 μl of 0.25M EDTA is added andincubation continued for 15 minutes. Lysis is brought about osmoticallyby diluting the samples to 400 μl with water. This procedure producesviable cell counts of 80-100 per ml.

When a 25 μl aliquot of this lysate is added into the assay reactionmix, the level of incorporation of ¹⁴ C-leucine into newly synthesisedprotein is .sup.˜ 10% of the blank without lysate. The result clearlyshowed that a minimum 16-fold dilution was necessary to reduce theeffect of the lysate to equal that of the blank.

In order to be as confident as possible that lysis of E.coli and E.colilysates would not compromise ribotoxin toxicity, 2 control assays wereperformed. The first added ribotoxin to a 16X diluted E.coli cell pelletso as to give a final concentration of 8 ng/ml in the assay mix aftercell lysis. Both these controls showed no deleterious affect from thelysates or the lysis procedure on the inhibitory action of ribotoxin.

These techniques were used to verify the synthesis of biologicallyactive protein analogue.

8.3 Bioactivity of Purified Restrictocin Analogues

    ______________________________________                                        Analogue  mean IC50 (M × 10E-11)                                        ______________________________________                                        TR1       41.1             s.d 14.3 (n = 6)                                   TR3       10.5             s.d 4.7 (n = 7)                                    TR4       10.8             s.d. 4.5 (n = 6)                                   TR5       13.4             s.d. 6.2 (n = 6)                                   TR6       4.95             s.d. 0.68 (n = 4)                                  Recombinant                                                                             2.27             s.d 1.04 (n = 6)                                   Restrictocin                                                                  ______________________________________                                    

Each IC50 was calculated from a line defined by triplicates of 5dilutions of ribotoxin wherein the calculated IC50 was greater than atleast 1 of the dilutions and also less than at least 1 of the dilutions.That is to say the IC50 was calculated from a line defined by the 5dilutions without recourse to extrapolation.

The figures for the specific activity of analogues TR5 and TR6 werecorrected for 10% and 15% contaminating protein respectively in thepurified product assuming that the contaminating protein had no activityin the protein synthesis assay. Other analogues tested contained nosignificant contamination. Restrictocin analogues TR5 and TR6 wereseparated from contaminants by reverse phase HPLC analysis andabsorbance of the eluate monitored at 214 nm. Resulting peaks wereidentified by N-terminal sequencing. Percentage contamination wascalculated by measurement of areas under the respective peaks.

Thus analogues TR1, TR3, TR4, TR5 and TR6 retain 6%, 22%, 21%, 17% and46% of the potency of native sequence recombinantly producedrestrictocin respectively. However this test is not conclusive of theutility of the protein analogues since the in vitro cytotoxicity assaydocumented below is used to test utility as an immunotoxin conjugate.For example the potency of analogue TR1 relative to native recombinantrestrictocin is significantly improved in the in vitro cytotoxicitytest.

EXAMPLE 9 Cytotoxicity of Immunotoxin

9.1 In vitro Cytotoxicity

This test shows the in vitro cytotoxicity of the immunotoxin against ahuman colo-rectal tumour cell line (Colo 205-ATCC No. CCL 222) whenribotoxin is conjugated to an antibody which recognises internalisableantigen on the cell line.

Colo 205 cells are grown in suspension in RPMI1640 medium with 2.0g/liter sodium bicarbonate, without glutamine, with 5% heat inactivatedfoetal calf serum, 2 mM L-glutamine and 50 μg/ml gentamicin. The cellsare trypsinised for 15 hours prior to harvesting and washing to removetrypsin. Cells are counted using haemocytometer blocks. For proteinsynthesis inhibition assays, cells are plated at 2-5×10⁴ cells/well in96 well plates in a volume of 100 μl. Immunotoxin samples are added to 4replicate wells. Immunotoxin is added at 12 doubling dilutions from2,000 to 0.98 ng/ml. 100 μl of culture medium is added to some wells ascontrol. Each plate is incubated for 24 hours at 37° C., prior to 2 μCiof ³ H-L-Leucine being added to each well. The plates are incubated at37° C. for a further 24 hours. 50 μl of trypsin is added to each welland the plates incubated for a further 15-20 minutes. The cells areharvested from each well/plate onto glass fibre mats and radioactivitydetermined using an betaplate scintillation counter such as supplied byLKB. Protein synthesis inhibition curves are constructed and IC₅₀ valuesgenerated (see below).

    ______________________________________                                        mean IC50 (M × 10E-11)                                                            analogue-19.9 Ab                                                              immunoconjugate                                                                           analogue alone                                          ______________________________________                                        TR1         206           58300                                               TR3         55.1          23300                                               TR4         77.5          49800                                               TR5         85.9          29700                                               TR6         20.2          21700                                               control     greater than 100,000                                              19.9 Ab alone                                                                 ______________________________________                                    

Thus analogues TR1, TR3, TR4, TR5 and TR6 are useful for preparation ofpotent immunotoxins.

9.2 In vivo Cytotoxicity

This test shows the in vivo cytotoxicity of the immunotoxin against thehuman tumour cell line, COLO 205, grown as subcutaneous xenografts inathymic mice. Control groups, utilising antibody alone or phosphatebuffered saline were also tested.

5×10⁶ COLO 205 cells are injected at a single sub-cutaneous site in theflanks of athymic mice. Tumours are allowed to grow and measured every3-4 days in two dimensions using calipers. Injection of test materialwas not initiated until tumours had reached 0.7-1.0 cm square. Thisstage is day 0 and test materials are injected intravenously into tailveins on day 0, 1 and 2. Tumour size continued the measured in twodimensions and presented as relative tumour volume. Measurements weremade every 3-4 days until the experiment was terminated.

This test compares the effects of phosphate buffered saline, antibodyalone (1.2 mg/kg) and the immunotoxin (preparation described above) (2.0mg/kg) on rumour growth.

The immunotoxin may give rise to a reduction in tumour size in contrastto the phosphate buffered saline (PBS) control and antibody alone.

EXAMPLE 10 Composition

The following illustrates a representative pharmaceutical dosage formcontaining an immunoconjugate (immunotoxin) of the present inventionwhich may be used for therapeutic purposes in humans.

Injectable Solution

A sterile aqueous solution, for injection, containing:

    ______________________________________                                        Restrictocin analogue/tumour                                                                        1.0    mg                                               selective antibody                                                            Sodium acetate trihydrate                                                                           6.8    mg                                               Sodium chloride       7.2    mg                                               Tween 20              0.05   mg per ml                                                                     of solution                                      ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 48                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CCCTCAGCTGCAGCTAC TTGGACTTGYATCAAYCARCA38                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CACCGACGTCGACTATTATTARTGRSWRCA CAGNCGCAGRTCRCCYTGRTT51                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AATTCGAGCTCGCCCGGGGATCCTGCAGCTACTTGGACTTG 41                                  (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCAAGCTTGGGTTGCAGGTCGACTATTATTAGTGGCTACACAGTC 45                              (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GTTTTCCCAGTCACGAC17                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CAGGAAACAGCTATGAC17                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              ( i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 17 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ACGGGAATGGCAAGCTC17                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GGTCTGGCTGTGCTTCG17                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 bases                                                           (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GAACCAGTGCGGGTAGC17                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 456 bases                                                         (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCTACTTGGACTTGTATCAACCAACAGCTGAATCCCAAGACAAACAAA48                            AlaThrTrpThrCysIleAsnGlnGlnLeuAsnProLysThrAsnLys                               151015                                                                       TGGGAAGACAAGCGGCTTCTATACAGTCAAGCCAAAGCCGAAAGCAAC96                            TrpGluAspLysArgLeuLeuTyrSerGlnAlaLysAlaGluSer Asn                             202530                                                                        TCCCACCACGCACCTCTTTCCGACGGCAAGACCGGTAGCAGCTACCCG144                           SerHisHisAlaProLeuSerAspGlyLysThrGlySerSe rTyrPro                             354045                                                                        CACTGGTTCACTAACGGCTACGACGGGAATGGCAAGCTCATCAAGGGT192                           HisTrpPheThrAsnGlyTyrAspGlyAsnGlyLysLeu IleLysGly                             505560                                                                        CGCACGCCCATCAAATTCGGAAAAGCCGACTGTGACCGTCCCCCGAAG240                           ArgThrProIleLysPheGlyLysAlaAspCysAspArgPr oProLys                             65707580                                                                      CACAGCCAGAACGGCATGGGCAAGGATGACCACTACCTGCTGGAGTTC288                           HisSerGlnAsnGlyMetGlyLysAspAsp HisTyrLeuLeuGluPhe                             859095                                                                        CCGACTTTTCCAGATGGCCACGACTATAAGTTTGACTCGAAGAAACCC336                           ProThrPheProAspGlyHisAs pTyrLysPheAspSerLysLysPro                             100105110                                                                     AAGGAAGACCCGGGCCCAGCGAGGGTCATCTATACTTATCCCAACAAG384                           LysGluAspProGlyPro AlaArgValIleTyrThrTyrProAsnLys                             115120125                                                                     GTGTTTTGCGGCATTGTGGCCCATCAGCGGGGGAATCAAGGCGATCTG432                           ValPheCysGlyIle ValAlaHisGlnArgGlyAsnGlnGlyAspLeu                             130135140                                                                     CGACTGTGTAGCCACTAATAATAG456                                                   ArgLeuCysSerHis                                                                145                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CAATCTAGAGGGTATTAATAATGTTCCCATTGGAGGATGATTAAATGGTAC51                         (2 ) INFORMATION FOR SEQ ID NO:12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CATTTAATCATCCTCCAATGGGAACATTATTAATACCCTCTAGATTGAGCT51                         (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCGAAGCACTGCCAGAACGGC21                                                       (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CACGACTATTGCTTTGACTCG21                                                       (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                           (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CTGAATCCCTGCACAAACAAA21                                                       (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CTGTGTAGCCACTGCGGTTAATAATAGTCG30                                              (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GCAGTTACTTGGACTTGCCTGAAC24                                                    (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GTTTCGTATTTGTTAGTTTTCGGGTTTTTCTGGTCGTTCAGGCAAGTCCAAGTAACTGCT60                GCA63                                                                         (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 bases                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GTCCGCCGAAACACTGCAAAGACGGTAACG30                                              (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GGTCACGACTACTGTTTCGACTCTAAAAAA30                                              (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B ) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      ACCAGAAAAACCCGTGTACTAACAAATACG30                                              (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      CTGTGCTCTCACTGCGGTTGATGGATCCCC30                                              (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GTAAAACTGGTTCCTCTTACCCGCACTGGTTCACTAACGGTTACGACGGT50                          (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                     AGTGAACCAGTGCGGGTAAGAGGAACCAGTTTTACCGTCAGACAGCGGAG50                          (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                       GATCCGTCGACTCAGTGAGAGCACAGTTTCAGTTCACCCTGGTTTTCTTTAGTGT55                    (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      AGGGTGAA CTGAAACTGTGCTCTCACTGAGTCGACG36                                       (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TTTGACTCGTGCA AACCCAAG21                                                      (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      ACAAATTCGACTCTTGTAAACCGAAA GAAA30                                             (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GGATCCTGCAGCTACTTGGACT 22                                                     (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      AlaThrTrpThr                                                                  (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      CTGTGTAGCCACTAATAATAGTCGAC26                                                  (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      LeuCysSerHis                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 149 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      AlaThrTrpThrCysIleAsnGlnGlnLeuAsnProLysThrAsnLys                              151015                                                                        TrpGluAspLysArgLeuLeuTyr AsnGlnAlaLysAlaGluSerAsn                             202530                                                                        SerHisHisAlaProLeuSerAspGlyLysThrGlySerSerTyrPro                              3540 45                                                                       HisTrpPheThrAsnGlyTyrAspGlyAsnGlyLysLeuIleLysGly                              505560                                                                        ArgThrProIleLysPheGlyLysAlaAspCysAsp ArgProProLys                             65707580                                                                      HisSerGlnAsnGlyMetGlyLysAspAspHisTyrLeuLeuGluPhe                              8590 95                                                                       ProThrPheProAspGlyHisAspTyrLysPheAspSerLysLysPro                              100105110                                                                     LysGluAsnProGlyProAlaArgValIle TyrThrTyrProAsnLys                             115120125                                                                     ValPheCysGlyIleValAlaHisGlnArgGlyAsnGlnGlyAspLeu                              130135 140                                                                    ArgLeuCysSerHis                                                               145                                                                           (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 150 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AlaValThrTrpThrCysLeuAsnAspGln LysAsnProLysThrAsn                             151015                                                                        LysTyrGluThrLysArgLeuLeuTyrAsnGlnAsnLysAlaGluSer                              202 530                                                                       AsnSerHisHisAlaProLeuSerAspGlyLysThrGlySerSerTyr                              354045                                                                        ProHisTrpPheThrAsnGlyTyrAspGlyAsp GlyLysLeuProLys                             505560                                                                        GlyArgThrProIleLysPheGlyLysSerAspCysAspArgProPro                              657075 80                                                                     LysHisSerLysAspGlyAsnGlyLysThrAspHisTyrLeuLeuGlu                              859095                                                                        PheProThrPheProAspGlyHisAspTyrLys PheAspSerLysLys                             100105110                                                                     ProLysGluAsnProGlyProAlaArgValIleTyrThrTyrProAsn                              115120 125                                                                    LysValPheCysGlyIleIleAlaHisThrLysGluAsnGlnGlyGlu                              130135140                                                                     LeuLysLeuCysSerHis                                                            145150                                                                    

We claim:
 1. A conserved protein analogue of a native ribotoxin in whichboth the protein analogue and the native ribotoxin can cleave only asingle phosphodiester bond of 28S rRNA in a 60S ribosomal subunit fromrat liver and in which the protein analogue contains only one cysteineavailable for covalent linkage to a partner said cysteine not beingpresent in the native ribotoxinwherein said peptide is selected from thegroup consisting of: (a) a restrictocin analogue selected from the groupconsisting of:i) a restrictocin analogue in which any one of thefollowing residues in native restrictocin (SEQ ID NO:10) has beensubstituted by cysteine: Lys 13, Lys 20, Lys 28, Lys 60, Lys 63, Lys 69,Ser 82, Lys 88, Lys 106, Lys 110 or Lys 128; ii) native restrictocin(SEQ ID NO:10) having additionally a C-terminus extension Cys 150-Gly151 attached thereto; and iii) native restrictocin (SEQ ID NO:10) havingadditionally an N-terminus extension Gly-Cys attached thereto; (b) amitogillin analogue selected from the group consisting of:i) amitogillin analogue in which any one of the following residues in nativemitogillin (SEQ ID NO:47) has been substituted by cysteine: Lys 13, Lys20, Lys 28, Lys 60, Lys 63, Lys 69, Ser 82, Lys 88, Lys 106, Lys 110 orLys 128; ii) native mitogillin .(SEQ ID NO:47) having additionally aC-terminus extension Cys 150-Gly 151 attached thereto; and iii) nativemitogillin (SEQ ID NO:47) having additionally an N-terminus extensionGly-Cys attached thereto; and (c) an alpha-sarcin analogue selected fromthe group consisting of:i) an alpha-sarcin analogue in which any one ofthe following residues in native alpha-sarcin (SEQ ID NO:48) has beensubstituted by cysteine: Lys 14, Lys 21, Lys 29, Lys 61, Lys 64, Lys 70,Ser 83, Lys 89, Lys 107, Lys 111 or Lys 129; ii) native alpha-sarcin(SEQ ID NO:48) having additionally a C-terminus extension Cys 151-Gly152 attached thereto; and iii) native alpha-sarcin (SEQ ID NO:48) havingadditionally an N-terminus extension Gly-Cys attached thereto.
 2. Aconserved protein analogue of a native ribotoxin in which both theprotein analogue and the native ribotoxin can cleave only a singlephosphodiester bond of 28S rRNA in a 60S ribosomal subunit from ratliver and in which the protein analogue contains only one cysteineavailable for covalent linkage to a partner said cysteine not beingpresent in the native ribotoxinwherein said protein is selected from thegroup consisting of: (a) a restrictocin analogue selected from the groupconsisting of:i) restrictocin analogues in which any one of thefollowing residues in native restrictocin (SEQ ID NO:10) has beensubstituted by cysteine: Lys 13, Ser 82, Lys 106 or Lys 110; and ii)native restrictocin (SEQ ID NO:10) having additionally a C-terminusextension Cys 150-Gly 151 attached thereto; (b) a mitogillin analogueselected from the group consisting of:i) a mitogillin analogue in whichany one of the following residues in native mitogillin (SEQ ID NO:47)has been substituted by cysteine: Lys 13, Ser 82, Lys 106 or Lys 110;and ii) native mitogillin (SEQ ID NO:47), having additionally aC-terminus extension Cys 150-Gly 151 attached thereto; and (c) analpha-sarcin analogue selected from the group consisting of:i) analpha-sarcin analogue in which any one of the following residues innative alpha-sarcin (SEQ ID NO:48) has been substituted by cysteine: Lys14, Ser 83, Lys 107 or Lys 111; and ii) native alpha-sarcin (SEQ IDNO:48) having additionally a C-terminus extension Cys 151-Gly 152attached thereto.
 3. A polynucleotide sequence that encodes a proteinanalogue as defined in claim 1 or
 2. 4. A replicative cloning vectorcomprising the polynucleotide sequence defined in claim
 3. 5. Areplicative expression vector comprising the polynucleotide sequencedefined in claim 3 operably linked to an expression control sequence. 6.Recombinant host cells transformed with a replicative expression vectordefined in claim 5.